The family of Reoviridae comprises a structurally related group of viruses. Members of this family of viruses can cause infections of the gastrointestinal system and the respiratory tracts in mammals and birds. Some viruses of this family can also infect plants.
The Reoviridae are non-enveloped, double-stranded RNA viruses made up of an icosahedral capsid that is typically composed of an outer protein layer and one or more inner protein layers. The genus Rotavirus forms part of the Reoviridae family. Rotaviruses form triple-layered virus particles. The outer layer of a rotavirus particle consists of a shell protein, VP7, and a spike protein, VP4; the middle layer consists of VP6; and the inner layer is formed by VP2. The triple-layered virus particle is infectious, after activation by trypsin. During rotavirus cell entry, the outer layer of the virus particles is removed by a mechanism dubbed “uncoating,” and the double-layered particle enters the cytoplasm. During cell entry, rotavirus penetrates a cellular membrane, possibly an endosomal membrane. The double-layered rotavirus particle is transcriptionally active. In the cytoplasm, it transcribes the genome, extruding mRNA through pores in its surface. The new transcripts are used to make rotavirus proteins and new double-stranded RNA genome segments, which are packaged into newly formed viral particles.
For some Reoviridae, the uncoating process can be simulated in vitro using purified viral particles. In the case of rotaviruses, calcium chelation or heat shock causes uncoating of viral particles in vitro. Once the outer protein layer has been removed, the resulting rotavirus double-layered particles can be “recoated” with recombinantly expressed versions of the proteins that form the outer layer. By recoating with recombinant VP4 and VP7, infectious recoated rotavirus particles that closely resemble mature virions can be formed. By recoating with VP7 alone, triple-layered, spike-less particles, which are minimally infectious, can be formed. By genetically modifying the coding regions of individual proteins of the outer layer and expressing them recombinantly, the properties of the proteins making up the outer layer can be studied in recoating experiments without requiring the production of fully recombinant viruses. To describe these types of experiments, the term “recoating genetics” has been coined.
In the case of reoviruses, the metastable state of the infectious subviral particle has allowed the use of cryo-electron microscopy (cryo-EM) in elucidating the structural basis for the priming mechanism that is used by reoviruses to enter their host cells. Similarly, in vitro recoated rotavirus particles have been used to study the molecular interactions in rotavirus assembly and uncoating using cryo-EM. The present invention builds on the technological advances that were brought about by these recoating and cryo-EM studies.